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Ezup Column Animal Genomic DNA Purification Kit
CAT. NO.: mB004-S, mB004-L
Package: 50/100 Preps
Kit Contents
Components
mB004-S, 50 Preps
mB004-L, 100 Preps
Ezup Column & Collection Tube
50
100
Buffer ACL
10 ml
20 ml
Buffer CL
12 ml
24 ml
CW1 Solution (concentrate)
13 ml
26 ml
CW2 Solution (concentrate)
9 ml
18 ml
CE Buffer (pH 9.0)
15 ml
30 ml
Proteinase K
1.2 ml
2.4 ml
Protocol
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Storage and stability
Ezup spin columns and all buffers should be stored dry, at room temperature (15-25°C), stable till see package.
Proteinase K solution is stable at room temperature for 6 months, aliquot to small amounts and store at -20°C for a long
term storage.
Safety instructions
Buffer ACL and Buffer CL are harmful in contact with skin if swallowed, please avoid contact with eyes, skin, and
clothes. Wash thoroughly after handling and see doctor if necessary.
24-hour emergency information
Emergency medical information in English, French, and German can be obtained 24 hours a day from: Poison
Information Center Mainz, Germany. Tel.: +49-(0)6131-19240
Introduction
Ezup Column Animal Genomic DNA Purification Kit is designed for rapid purification of genomic DNA (but other kinds
of DNA, e.g. mitochondrial DNA will be also copurified) from fresh or frozen animal tissues.
Samples are first lysed using proteinase K in an optimized buffer. Then the lysate is loaded onto the Ezup spin column,
DNA is selectively bound to the Ezup membrane embed in the column in the presence of high concentrations of chaotropic
salt. During wash steps, protein and other impurities were removed and DNA is then eluted in water or low-salt buffer.
Purified DNA typically has an A260/A280 ratios of 1.7-1.9, and is highly suited for most downstream applications such as
Updated:06/2014
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PCR, Southern blotting, RAPD and RFLP.
The purification procedure requires no phenol or chloroform extraction or alcohol precipitation, and involves minimal
handling. The whole procedure takes only 20 minutes after sample preparation.
Yield of DNA varies between samples depending on the amount and type of tissue, for most tissues 5-10 µg DNA can
be obtained from 25 mg materials.
Features
1.
High quality of DNA, OD260/OD280 of purified DNA is generally 1.7~1.9.
2.
Fast and effective. Fast and easy processing using a rapid spin-column format.
3.
Compatible with many downstream applications such as PCR, restriction digestion and hybridization. .
4.
No phenol/chloroform extraction or ethanol precipitation is required.
Materials Supplied by User
Microcentrifuge capable of at least 12,000 × g
Pipets and pipet tips
Vortexer
Ethanol (96-100%)
RNase A (100 mg/ml, Optional for RNA-free DNA)
Microcentrifuge tubes (1.5 ml or 2 ml)
Water bath for heating at 56°C
Before Starting
This protocol is designed for purification of total DNA from animal tissues, including rodent tails. All centrifugation steps
are carried out at room temperature (15-25°C) in a microcentrifuge. It is strongly advised that you read this booklet
throughly before starting. Ezup Column Animal Genomic DNA Purification Kit is designed to be simple, fast and reliable
provided that all steps are followed diligently. Prepare all components, and have the necessary materials as outlined before
starting.
Proteinase K is supplied in a ready-to-use solution form, but RNase A is not provided in this kit, if RNA-free DNA are
required, please prepare RNA solution and see protocol to add the RNA removal step.
Check the Buffer ACL and Buffer CL for salt precipitation before each use. If necessary, redissolve the precipitate by
warming the solution at 56°C, then cool back down to room temperature before use.
CE Buffer is 10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0. Water can be used as eluate in the final step if EDTA should be
avoided for the following applications, but it is not recommended if the pH of water is less than 7.0.
CW1 Solution and CW2 Solution are supplied as concentrates. Before using for the first time, add 17 ml ethanol to 13
ml CW1 Solution, 21 ml ethanol to 9 ml CW2 Solution, respectively.
Preheat the water bath or rocking platform to 56°C.
Procedures
1.
Cut up to 25 mg tissue into small pieces, ground in in liquid nitrogen, transfer the powder to a 1.5 ml
microcentrifuge tube and add 180 µl Buffer ACL, mix well.
Note 1: Don't use more than 25 mg tissue, exceed starting materials will lead to lower yield.
Note 2: For frozen tissue, avoid repeated thawing and freezing of samples since this will lead to DNA degradation.
Note 3: For rodent tails, take 0.4~0.6 cm lengths of tail for rat and 1.0~1.2 cm for mouse.
Note 4: For tissues with a very high number of cells, such as spleen, no more than 10 mg starting material should be used.
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2.
Add 20 µl proteinase K. Mix thoroughly by vortexing, and incubate at 56°C until the tissue is completely lysed.
Note 1: Vortex occasionally during incubation to disperse the sample.
Note 2: Lysis time varies depending on the type of tissue, typically 1~3 h, or can be lysed overnight.
Note 3: After incubation the lysate may appear viscous, but should not be gelatinous as it may clog the Ezup column. If the
lysate appears very gelatinous, longer incubation is required.
Note 4: If RNA-free genomic DNA is required, add 4 µl RNase A (100 mg/ml), mix by vortexing, and incubate for 2 min at
room temperature before continuing with step 3.
3.
Vortex for 15s. Add 200 µl Buffer CL to the sample, and mix thoroughly by vortexing. Then add 200 µl ethanol
(96-100%), and mix again thoroughly.
4.
Transfer the mixture from step 3 (including any precipitate) into the Ezup column placed in a 2 ml collection
tube. Centrifuge at 9,000 x g (12,000 rpm) for 1 min. Discard the flow-through.
5.
Add 500 µl CW1 Solution, and centrifuge for 1 min at 9,000 x g (12,000 rpm). Discard the flow-through.
Note: Check the label to ensure CW1 Solution was diluted with ethanol.
6.
Add 500 µl CW2 Solution, and centrifuge for 1 min at 9,000 x g (12,000 rpm). Discard the flow-through.
Note: Check the label to ensure CW2 Solution was diluted with ethanol.
7.
Place the empty column in the microcentrifuge and centrifuge for an additional 2 min at 9,000 x g (12,000 rpm)
to dry the Ezup membrane. Discard flow-through and transfer the spin column to a clean 1.5 ml centrifuge
tube.
Note: It is important to dry the membrane of the Ezup spin column, since residual ethanol may interfere with subsequent
reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution.
8.
Add 50-100 µl Buffer CE directly onto the center part of Ezup membrane. Incubate at room temperature for 1
min, and then centrifuge for 1 min at 9,000 x g (12,000 rpm) to elute the DNA.
Note 1: Warm the Buffer CE to 60°C will increase the elution efficiency.
Note 2: Elution with more than 100 µl (e.g. 200 µl) increases the DNA yield, but the concentration will be lower.
Note 3: For maximum DNA yield, repeat elution once as described in this step.
Note 4: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first eluate.
Note 5: For maximun DNA concentration, use the eluate in the microcentrifuge tube for the second elution step.
Updated:06/2014
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